Human Embryonic Stem Cells and their Derivation
Embryonic stem cells are undifferentiated cells that can grow in in vitro culture conditions, while retaining their capability to differentiate into specialized cell types having particular functions. Over the last few decades there has been a great interest in isolation and culture of human embryonic stem cells (hES-cells), as such cells can potentially provide a supply of readily available differentiated cells and tissues of all types to be used for therapeutic purposes in cell transplantation and gene therapy in humans, as well as for other medical uses such as drug discovery.
Early work on embryonic stem cells was done in mice (reviewed in Robertson, 1997; Pedersen, 1994). Current definition of both mouse and human embryonic stem cells includes the requirements for them to be capable of indefinite proliferation in vitro in an undifferentiated state, to retain a normal karyotype and to retain the potential to differentiate to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm).
Methods for isolating and growing primordial stem cells (consisting of stem cells derived from both inner cell masses of embryos and from germ-cells of fetuses) from non-human primates and humans have been described previously in several publications (reviewed by Pedersen, 1999). A few of the seminal publications in this area are Thomson et al. (1995) (describing procedures for isolating Rhesus monkey primordial stem cells) and Thomson at al. (1998) (describing procedures for isolating human embryonic stem cell lines). Production of a new hES-cell line is called derivation. Once the population of undifferentiated cells has been derived from one embryo, they can be propagated indefinitely to yield unlimited numbers of cells that can be frozen and thawed or continuously cultured for several hundreds of cell divisions without the cells losing their ability to grow. At this stage it is called cell line. However, in order to maintain the undifferentiated state in culture, the calls need to be selected and passaged frequently to avoid the spontaneous differentiation otherwise taking place in growing colonies.
The source of hES-cell lines is excess human embryos donated for research by couples that have completed their fertility treatment and no longer need their embryos. In addition, embryos deemed to be unsuitable for clinical use due to poor viability or identified abnormalities, or even embryos specifically created for this purpose (under certain legislations) can be used. At a blastocyst stage, 5 or 6 days after fertilisation, the embryo consists of two distinctive cell populations; inner cell mass (ICM) cells and trophectodermal (TE) cells. Derivation of new hES-cell lines is based on culturing whole ICMs or cells thereof on a feeder cell layer. These cells are usually isolated from surrounding TE-cells by immunosurgery, although whole embryos can also be directly plated on feeder cells, whereby ICM-cells emerge from surrounding TE-cells during the course of culture. Earlier stage embryos can also be used, but the underlying principle remains the same: stem cells are derived from pluripotent undifferentiated cells within an embryo. The efficiency of hES-cell line derivation varies vastly between different laboratories, depending on isolation conditions, experience of the group and the quality of the embryos used. However, as the number of embryos donated for this purpose is usually limited, little experimentation has been conducted in this area and groups tend to use the methods originally employed with perhaps only slight variations. The success rates (percentage of successfully derived hES-cell lines per embryo used) vary from as low as 5% (Cowan et al. 2004) to 50% (Reubinoff et al. 2000). However, the comparison of success rates between groups is quite difficult because of the differences with respect to how the groups report their data; per cleavage-stage embryo or per blastocyst used, per total number of embryos entered into the program or per successfully plated blastocyst or ICM and so-forth.
So far the concerns about hES-cell line derivation efficiency have been mainly due to attempts to minimize the number of embryos needed. However, recent attention has been directed towards derivation of hES-cells from particular, individual embryos, for example of those identified by preimplantation genetic testing or diagnosis (together, “PGD”) to carry a specific genetic defect or chromosomal abnormality (Amit et al. 2004b; Galat et al. 2004; Verlinsky et al. 2004). Also the use of somatic cell nuclear transfer techniques for generating embryos for stem cell derivation (Hwang et al. 2004, 2005) puts more emphasis on the need to optimize the efficiency of successful hES-cell line derivation.
Most of the reported hESC-derivations have been performed with blastocysts grown in routine clinical conditions, in a variety of different embryo culture media. As>90% of the IVF clinics, especially in United States, use standard cell culture atmosphere of 5% CO2 in air for culture, it is safe to assume that by far most hESC-derivations have been done with embryos produced in these conditions.
Production of Embryo Outgrowths and Other Undefined Embryo-Derived Cell Lines
Currently it is not possible to culture mammalian embryos in in vitro conditions much beyond the advanced blastocyst stage (in humans until day 8 or 9 of development) without embryos losing their identity as an embryo and their subsequent developmental potential to form a fetus. However, embryos or embryonic cells cultured for extended periods in vitro do have the capability to divide and grow into undefined cell populations (Hogan et al. 1994; Flechon et al. 1995; van Stekelenburg-Hamers et al. 1995; Tanaka et al. 1998; Talbot et al 2000; Leoni et al. 2000; Shimada et al. 2001; AlBadr & Handyside 2003). The acquisition of this kind of embryo-derived cell lines or outgrowths has important applications for diagnostic and research applications in the general field of in vitro fertilization and related assisted reproductive technologies (ART). The major restriction of any PGD-analysis is the limited number of cells available for analysis, combined with the uncertainty about how representative those few cells are for the embryo as a whole. Clinically and diagnostically, PGD analysis able to be performed on an unlimited number of cells derived from one embryo would offer improved accuracy and/or greater flexibility over the current analysis methods. A significant research application for such embryo-derived cells would be to use them for re-analysis in the course of development of new PGD-analysis methods, providing thereby the possibility to confirm an analysis made with a new method involving one or a few cells with a proven and tested method that requires many cells, and thereby constituting a valuable tool for refining PGD-methods.
Although there have been a few reports detailing derivation of trophectodermal or other, undefined embryo-derived cell lines from animal embryos (Hogan et al, 1994; Flechon et al. 1995; van Stekelenburg-Hamers et al. 1995; Tanaka et al. 1998; Talbot et al 2000; Leoni et al. 2000; Shimada et al. 2001; AlBadr & Handyside 2003), no successful derivations of non-pluripotent cell lines (such as trophoblast stem cell lines) have been reported from human embryos, and neither pluripotent nor non-pluripotent cell lines have been successfully derived from embryo biopsies, despite some attempts (Geber et al. 1995; Geber & Sampaio 1999). Recent success in producing embryonic stem cell line from a biopsied blastomere in mouse (Chung at al. 2005) suggests that in certain cases this may be possible, but the methods used involve aggregation of blastomere with an existing stem cell line, thus creating mosaic cell lines. Whether these methods would be applicable in generating human embryonic stem cell lines is not yet known. Nevertheless, due to complications owing to mosaicism, these methods may not be readily suitable in clinical situations for evaluation of particular embryos.
Culture of Human Embryonic Stem Cell Lines
The usual method for “maintenance” culture of human embryonic stem cells after the initial derivation phase is to grow them on a layer of somatic feeder cells, such as foetal fibroblasts. Cells can be passaged (moved from an old feeder cell dish to a now dish) manually by cutting colonies into small pieces by a blade or glass pipette, teasing the fragments away from the bottom of the dish and transferring them to a new dish (“mechanical” or “manual passaging”). Another method of passaging involves the use of enzymes or enzyme-free buffer solutions to disperse colonies into cell clumps or a single cell suspension and transfer all or some of these cells into new dishes (“bulk passaging”). It is also possible to grow hES-cells without feeder cells on dishes coated with gelatin or extracellular matrix and using either media conditioned by feeder cells or a defined media with exogenous growth factors (Xu, C. et al. 2001, Nat Biotechnol 19:971; Bodnar et al., 2004, U.S. Pat. No. 6,800,480).
The requirement for passaging of hES-cells is not driven only by the need to chance them to a fresh batch of feeder cells, but due to their propensity to spontaneously differentiate if the colonies are left to grow too large. Even in feeder free conditions regular passage is required to prevent differentiation. Maintaining stem cells in undifferentiated stage is controlled by a complicated network of signal transduction pathways, which in turn are affected by culture conditions and cellular interactions within a colony of stem cells. The presence of even small numbers of differentiated cells and their interaction with neighbouring undifferentiated cells can play an important role in determining the fate of the adjacent cells.
The area of improvement that has received most attention lately has been the attempts to reduce reliance on animal-derived cells and any other animal or bacteria-derived biological products in culture, in favour of more defined culture conditions (Amit et al. 2004a). Not much progress has been made in the field of improving cell culture conditions in the more “traditional” culture systems (including culture on feeder cells).
Culture of Embryo Outgrowths and Undefined Embryo-Derived Cell Lines
Because only a few of the successful cases have been reported in the literature, and employing a variety of species, there is no such thing as “a routine culture method” for other embryo derived outgrowths and cell lines. Many of the approaches have utilized very similar culture conditions as used for stem cells, including the use of feeder cells (Tanaka et al. 1998; Talbot et al. 2000). However, feeder-free culture systems have also been used (Leoni et al. 2000). The best culture conditions have probably not yet been defined, but the culture on feeder cells still is the most used approach.
Spontaneous Differentiation of Human Embryonic Stem Cell Lines
Human embryonic stem cells in culture differentiate spontaneously after a prolonged culture or if cultured in sub-optimal culture conditions. The first recognisable sign of differentiation is the upregulation and expression of markers not present in undifferentiated populations. The exact type of markers depends on towards which cell lineage the differentiation is progressing (e.g. TDGF1, AP-2, MSX-2 (reviewed by Rao & Stice, 2004)
The Role of Oxygen Tension
All published initial derivations of human embryonic stem cells, trophectodermal stem cells and even all the attempts to grow embryo biopsies in order to generate embryo-derived cell types, have utilized the traditional cell culture gas atmosphere of approx. 5% CO2 in air. These same conditions have also been used universally for the subsequent culture of hES-cells. Likewise, most IVF clinics still rely on traditional high oxygen atmosphere in their embryos culture, although change to reduced oxygen culture is slowly gathering momentum especially in Australia and Europe.
Studies on embryos of various species have suggested that early pre-implantation embryo development is improved in an atmosphere of low oxygen, usually in a gas mixture of 5% CO2, 5% O2 and 90% N2 (Catt & Henman 2000; Orsi & Leese 2001). Although embryo-derived cell lines quickly lose their original “embryonic development program” (the genetic program controlling early embryo development), to be replaced by another type of developmental control, it is proposed herein that the two developmental patterns have enough in common to justify the hypothesis that the derivation and culture of embryo-derived cell lines (including, but not exclusive only to bona fide human embryonic stem cells) will be improved in low oxygen culture conditions. The process can be improved even further, as described herein, by utilizing a system where both the production of embryos (blastocysts or earlier stages), as well as derivation and subsequent culture of embryonic stem cell lines is performed in low oxygen culture conditions.
Thus there is still a need for novel methods of increasing the growth rate and/or reducing the spontaneously occurring differentiation of hES-cells in culture thereby increasing the potential for utilization of these cells
Thus, it is an object of the present invention to provide improved methods for derivation and/or culture of embryonic stem cells and cell lines as well as other types of embryo-derived cells and cell lines.